Digital PCR: Endless Frontier of 'Divide and Conquer'

Abstract

Digital polymerase chain reaction (PCR) is becoming ever more recognized amid the overwhelming revolution in DNA quantification, genomics, genetics, and diagnostics led by technologies such as next generation sequencing and studies at the single-cell level. The demand to quantify the amount of DNA and RNA has been driven to the molecular level and digital PCR, with its unprecedented quantification capability, is sure to shine in the coming era. Two decades ago, it emerged as a concept; yet one decade ago, integration with microfluidics invigorated this field. Today, many methods have come to public knowledge and applications surrounding digital PCR is mounting. However, to reach wider accessibility and better practicality, efforts are needed to tackle the remaining problems. This perspective looks back at several inspiring and influential digital PCR approaches in the past and tries to provide a futuristic picture of the trends of digital PCR technologies to come.

Keywords: digital polymerase chain reaction (PCR); emulsion droplet; microfluidics; microwell chip.

Figure 1

An overview of representative digital PCR approaches. For each partition shown herein represents approximately 1000 partitions in the corresponding methods. Their sizes too roughly reflect those in real-world situations. Micro-chambers in microfluidic chips are shown in pink and emulsion droplets in light blue. (a) Digital PCR in well plates [3,4]; (b) The first microfluidic chip for digital PCR in 2006 [5]; (c) SlipChip in 2010 [6]; (d) One-million droplet array in 2011 [7]; (e) Megapixel digital PCR in 2011 [8]; (f) Digital Array IFC (type: qdPCR 37k) from Fluidigm’s Biomark HD (Fluidigm Corporation, South San Francisco, CA, USA); (g) RainDrop digital PCR from Raindance Technologies (Lexington, MA, USA); (h) QuantStudio 3D from Life Technologies (now Thermo Fisher, Waltham, MA, USA); (i) QX200 droplet digital PCR platform from Bio-Rad (Hercules, CA, USA); (j) The densest and smallest microwell-based microfluidic digital PCR to date in 2012 [9]; (k) Chip-free digital PCR in droplets generated via micro-channel array (MiCA) centrifugation in 2017 [10].

Figure 2

Microfluidic digital PCR partitioning and counting strategies. (a) Emulsion droplet generation using flow-focusing microfluidic chip; (b) Serial droplet fluorescence reading; (c) Minute volumes of PCR sample are partitioned in micro-well microfluidic chip; (d) Planar imaging for fluorescence positive chamber counting.